Many human diseases result from complex interactions between genome and environmental agents. Mutation is a frequent consequence of unrepaired DNA damage. The nucleotide excision repair (NER) pathway plays a particularly important role in the repair of environmental mutagen-induced DNA damage. NER repairs a wide variety of helix-distorting `bulky' DNA lesions that result from damaging agents such as UV radiation, cisplatin or reactive oxygen species (ROS), as well as many chemicals found in cigarette smoke. There are two sub- pathways of NER, termed global genomic repair (GG-NER) and transcription-coupled repair (TC-NER). These pathways differ mainly in their recognition of damage. While an RNA polymerase blocked by a lesion initiates TC-NER, restricting it to the transcribed strand of active genes, GG-NER surveys the entire genome for distorting DNA lesions. Therefore, GG-NER requires specific proteins for damage recognition. Xeroderma pigmentosum complementation group C (XPC) and Xeroderma pigmentosum complementation group E (XPE or DDB2) are two important damage recognition factors that recognize DNA lesions during GG-NER. Ubiquitination plays crucial roles in both TC-NER and GG-NER. DDB2 and XPC are both covalently modified by ubiquitination during GG-NER. Although ubiquitination plays a crucial role in the regulation of DNA damage recognition by DDB2 and XPC, the sites of ubiquitin attachment in DDB2 and XPC and ubiquitin chain topologies remain unidentified. These pieces of information are needed to understand why ubiquitinated XPC is stable and XPC can be recycled after deubiquitination, while ubiquitinated DDB2 is labile and degraded by the proteasome. Incidentally, our preliminary studies show that two novel deubiquitinating enzymes (DUBs), USP24 and OTUD4, differentially regulate the steady state levels of XPC and DDB2 in the absence of UV irradiation. Time course experiments following UV irradiation also show increased levels of XPC ubiquitination after UV irradiation in OTUD4 depleted cells. Since XPC and DDB2 play roles in DNA damage recognition, we hypothesize that USP24 and OTUD4 regulate NER. The main objective of this study is to identify ubiquitin attachment sites in DDB2 and XPC, reveal ubiquitin linkage topologies that decide the fate of XPC and DDB2 and define the mechanisms by which USP24 and OTUD4 regulate XPC and DDB2 through their deubiquitinase activities. Our long-term goal is to understand how DNA damage is recognized and repaired in human cells. Knowledge about XPC and DDB2 ubiquitination and deubiquitination will help understand the dynamics of DNA damage recognition. The R21 funding mechanism will allow us to define the nature of XPC and DDB2 ubiquitination and investigate the novel roles of OTUD4 and USP24 in DNA repair. The following three Specific Aims are proposed to achieve our goal. In Aim 1, we will identify attachment sites and nature of XPC/DDB2 ubiquitination before and after UV irradiation. Aim 2 will define the mechanisms by which USP24 and OTUD4 control the levels of XPC and DDB2. In Aim 3 we will determine the physiological roles of USP24 and OTUD4 in DNA repair and genomic stability.